Patterns of morphological variation in enamel–dentin junction and outer enamel surface of human molars

Tooth crown patterning is governed by the growth and folding of the inner enamel epithelium (IEE) and the following enamel deposition forms outer enamel surface (OES). We hypothesized that overall dental crown shape and covariation structure are determined by processes that configurate shape at the enamel–dentine junction (EDJ), the developmental vestige of IEE. This this hypothesis was tested by comparing patterns of morphological variation between EDJ and OES in human permanent maxillary first molar (UM1) and deciduous second molar (um2). Using geometric morphometric methods, we described morphological variation and covariation between EDJ and OES, and evaluated the strength of two components of phenotypic variability, canalization and morphological integration, in addition to the relevant evolutionary flexibility, i.e. the ability to respond to selective pressure. The strength of covariation between EDJ and OES was greater in um2 than in UM1, and the way that multiple traits covary between EDJ and OES was different between these teeth. The variability analyses showed that EDJ had less shape variation and a higher level of morphological integration than OES, which indicated that canalization and morphological integration acted as developmental constraints. These tendencies were greater in UM1 than in um2. On the other hand, EDJ and OES had a comparable level of evolvability in these teeth. Amelogenesis could play a significant role in tooth shape and covariation structure, and its influence was not constant among teeth, which may be responsible for the differences in the rate and/or period of enamel formation.     

The fibrillar structure of cementum and dentin at the cemento-dentinal junction in rat molars

The cemento-dentinal junction was examined in demineralized rat molars with complete roots by scanning electron microscopy combined with NaOH maceration. It is established that the NaOH maceration removes interfibrillar substances and cells from connective tissues selectively without structural damage to collagen fibrils. The cementum was detached from the dentin by the maceration. The inner cementum surface facing the dentin and the outer dentin surface facing the cementum were observed. In acellular cementum, both the outer dentin surface and the inner cementum surface had a smooth appearance. There was little indication of fibrils intermingling between dentin and cementum. In contrast, both the inner cementum surface and outer dentin surface in cellular cementum had an uneven appearance due to the irregular arrangement of collagen fibrils. Point-like protrusions of fibril bundles were observed on both surfaces. Some (not all) of these point-like protrusions appeared to correspond to places of fibrillar intermingling between dentin and cementum.     

The enamel–dentine junction in the postcanine dentition of Australopithecus africanus: intra-individual metameric and antimeric variation

We used micro-computed tomography and virtual tools to study metric and morphological features at the enamel–dentine junction and on the outer enamel surface in the postcanine dentition of an exceptionally well-preserved maxilla and mandible of an early hominin. The fossil, Sts 52 from Sterkfontein, South Africa, is attributed to Australopithecus africanus and is about 2.5 million years old. For comparative purposes in this exploratory study, we also used micro-computed tomography to analyse the dentition of a common chimpanzee ( Pan troglodytes ), a pygmy chimpanzee ( Pan paniscus ) and three extant humans. Metameric variation of the 3D enamel–dentine junction in the two chimpanzee mandibles was much smaller than in extant humans. Variation in metameric shape was high and complex. Notably, the mandibular metameric variation in extant humans can be greater within individuals, as compared with variation between individuals, with differences in shape appearing greater for M2 compared with M1. We recommend the use of a new approach in which individual metameric variation is systematically assessed before making inferences about differences between fossil hominin species. The fossil hominin examined in this study showed a metameric pattern of mandibular variation in shape that was comparable to the pattern seen in two chimpanzees. This degree of metameric variation appeared relatively small compared with the much larger patterns of variation observed within and between extant humans.     

Comparative Anatomy,Evolution, and Homologies of Tetrapod Hindlimb Muscles,Comparison with Forelimb Muscles,and Deconstruction of the Forelimb‐Hindlimb Serial Homology Hypothesis

For more than two centuries, the idea that the forelimb and hindlimb are serially homologous structures has been accepted without serious question. This study presents the first detailed analysis of the evolution and homologies of all hindlimb muscles in representatives of each major tetrapod group and proposes a unifying nomenclature for these muscles. These data are compared with information obtained previously about the forelimb muscles of tetrapods and the muscles of other gnathostomes in order to address one of the most central and enigmatic questions in evolutionary and comparative anatomy: why are the pelvic and pectoral appendages of gnathostomes generally so similar to each other? An integrative analysis of the new myological data, combined with a review of recent paleontological, developmental, and genetic works and of older studies, does not support serial homology between the structures of these appendages. For instance, many of the strikingly similar forelimb and hindlimb muscles found in each major extant tetrapod taxon were acquired at different geological times and/or have different embryonic origins. These similar muscles are not serial homologues, but the result of evolutionary parallelism/convergence due to a complex interplay of ontogenetic, functional, topological, and phylogenetic constraints/factors. Anat Rec, 297:1047–1075, 2014. © 2014 Wiley Periodicals, Inc.     

The behavioral profile of children aged 1–5 years with sex chromosome trisomy (47,XXX, 47,XXY, 47,XYY)

Children with SCT have an increased risk of suboptimal neurodevelopment. Previous studies have shown an elevated risk for neurobehavioral problems in individuals with SCT. However, not much is known about neurobehavioral problems in very young children; knowledge that could help with early identification of children at risk for suboptimal development, and that could help establish targets for early intervention. This study addressed the question of what the behavioral profile of children with SCT aged 1–5 years looks like. In total, 182 children aged 1–5 years participated in this study (N_SCT=87, N_{nonclinical controls} = 95). Recruitment and assessment took place in the Netherlands and the United States. The SCT group was recruited through prospective follow‐up (50%), information seeking parents (31%), and clinical referral (18%). Behavioral profiles were assessed with the child behavior checklist and the ages‐and‐stages social–emotional questionnaire. Levels of parent‐rated problem behavior were higher in children with SCT. Difficulties with overall social–emotional functioning were already present in 1‐year‐olds, and elevated scores were persistent across the full age range. Affective and pervasive developmental behaviors were seen in late toddlerhood and prominent at preschool age. Anxiety, attention deficit, and oppositional defiant behaviors were seen in preschool‐aged children. Within this cross‐sectional study, the developmental trajectory of affective, pervasive developmental, and oppositional defiant behaviors seemed to be different for SCT children than nonclinical controls. Collectively, these results demonstrate the importance of behavioral screening for behavioral problems in routine clinical care for children with SCT from a young age. Social–emotional problems may require special attention, as these problems seem most prominent, showing increased risk across the full age range, and with these problems occurring regardless of the timing of diagnosis, and across all three SCT karyotypes.     

Association of rudimentary sacral zygapophyseal facets and accessory and ligamentous articulations: Implications for load transmission at the L5‐S1 junction

Weight transmitted from the fifth lumbar vertebrae to the sacrum is distributed as three separate components between (a) the vertebral bodies anteriorly, (b) the transverse elements intermediately, and (c) the lumbosacral facet joints, posteriorly. The posterior components of the fifth lumbar vertebra share greater proportion of load in comparison with the posterior elements of the upper lumbar vertebral levels. This study focuses on rudimentary lumbosacral facet articulations and their possible effects on load sharing at this region. Twenty sacra bearing rudimentary articulations were collected for analysis. Sixteen of these sacra presented unilateral rudimentary facets, and the remaining four had facets that were bilaterally rudimentary. Thirteen of the sacra with unilateral rudimentary facets showed an accessory articulating area on the upper surface of the ala on the same side as the rudimentary zygapophyseal facet. The remaining three sacra (out of the 16) showed evidence of strong ligamentous attachments between the L5 and S1 transverse elements on the sides of the rudimentary facets. All the sacra with bilateral rudimentary facets demonstrated bilateral accessory L5–S1 articulations. These observations indicated that load transmission at lumbosacral junctions bearing a rudimentary facet joint is not normal and that their associations with strong L5–S1 lumbosacral ligamentous attachments or accessory articulations at the transverse elements serve a compensatory mechanism for load sharing. Clin. Anat. 23:707–711, 2010. © 2010 Wiley‐Liss, Inc.     

Morphology,topology and dimensions of the heart and arteries of genetically normal and mutant mouse embryos at stages S21–S23

Accurate identification of abnormalities in the mouse embryo depends not only on comparisons with appropriate, developmental stage‐matched controls, but also on an appreciation of the range of anatomical variation that can be expected during normal development. Here we present a morphological, topological and metric analysis of the heart and arteries of mouse embryos harvested on embryonic day (E)14.5, based on digital volume data of whole embryos analysed by high‐resolution episcopic microscopy (HREM). By comparing data from 206 genetically normal embryos, we have analysed the range and frequency of normal anatomical variations in the heart and major arteries across Theiler stages S21–S23. Using this, we have identified abnormalities in these structures among 298 embryos from mutant mouse lines carrying embryonic lethal gene mutations produced for the Deciphering the Mechanisms of Developmental Disorders (DMDD) programme. We present examples of both commonly occurring abnormal phenotypes and novel pathologies that most likely alter haemodynamics in these genetically altered mouse embryos. Our findings offer a reference baseline for identifying accurately abnormalities of the heart and arteries in embryos that have largely completed organogenesis.     

Enamel hypoplasia in molars of sheep and goats, and its relationship to the pattern of tooth crown growth

Enamel is the most highly mineralized and durable tissue of the mammalian body. As enamel does not undergo remodeling or repair, disturbances of enamel formation leave a permanent record in the tissue that can be used for life history reconstruction. This study reports light and scanning electron microscope findings on hypoplastic enamel defects, and on the chronology of crown growth in the molars of sheep and goats. A marked reduction of enamel extension rates in cervical compared with more cuspal crown portions of sheep and goat molars was recorded, with formation of the cervical 25% of the crown taking about the same time as that of the upper 75% of the crown. This explains the more frequent occurrence of enamel hypoplasia in cervical compared with upper and middle crown portions. Regarding the identification of hypoplastic enamel defects by external inspection, our results suggest a dependence on the type of defect and the associated presence of smaller or larger amounts of coronal cementum. Defects considered to reflect a slight to moderate impairment of secretory ameloblast function can normally be correctly diagnosed as they are not occluded by thick layers of cementum. In contrast, defects denoting a severe impairment of enamel matrix secretion can typically not be correctly identified because they are occluded by large amounts of cementum, so that neither depth nor extension of the defects can be assessed on external inspection. In these cases, microscopic analysis of tooth sections is required for a correct diagnosis of the hypoplastic enamel defects.     

Retinoic acid controls expression of tissue remodeling genes Hmgn1 and Fgf18 at the digit–interdigit junction

Previous studies on retinoic acid receptor (RAR) mutants suggested that retinoic acid (RA) is required for loss of interdigital mesenchyme during digit formation. Here, we report that the RA‐generating enzyme retinaldehyde dehydrogenase‐2 (Raldh2) is expressed in the interdigital mesenchyme whereas Cyp26b1, controlling RA degradation, is expressed in digits, limiting autopodal RA action to the interdigital zones. Embryonic day 13.5 Raldh2?/? mouse embryos lose expression of the RARE‐lacZ RA‐reporter transgene and matrix metalloproteinase‐11 (Mmp11) throughout the interdigital mesenchyme, while expression of RARb, Fgf18, and high mobility group N1 (Hmgn1) is lost at the digit–interdigit junction. Raldh2?/? autopods exhibit reduced interdigital apoptosis associated with loss of Bmp7 expression, but Bmp2, Bmp4, Msx2, and Fgf8 were unaffected. Although interdigital expression of Hmgn1 was greatly down‐regulated in Raldh2?/? autopods, complementary expression of Sox9 in digit cartilage was unaffected. Regulation of Hmgn1 and Fgf18 at the digit–interdigit junction suggests RA controls tissue remodeling as well as apoptosis. Developmental Dynamics 239:665–671, 2010. © 2009 Wiley‐Liss, Inc.     

Growth‐related structural,biochemical, and mechanical properties of the functional bone–cartilage unit

Articular cartilage and subchondral bone act together, forming a unit as a weight‐bearing loading‐transmitting surface. A close interaction between both structures has been implicated during joint cartilage degeneration, but their coupling during normal growth and development is insufficiently understood. The purpose of the present study was to examine growth‐related changes of cartilage mechanical properties and to relate these changes to alterations in cartilage biochemical composition and subchondral bone structure. Tibiae and femora of both hindlimbs from 7‐ and 13‐week‐old (each n = 12) female Sprague‐Dawley rats were harvested. Samples were processed for structural, biochemical and mechanical analyses. Immunohistochemical staining and protein expression analyses of collagen II, collagen IX, COMP and matrilin‐3, histomorphometry of cartilage thickness and COMP staining height were performed. Furthermore, mechanical testing of articular cartilage and micro‐CT analysis of subchondral bone was conducted. Growth decreased cartilage thickness, paralleled by a functional condensation of the underlying subchondral bone due to enchondral ossification. Cartilage mechanical properties seem to be rather influenced by growth‐related changes in the assembly of major ECM proteins such as collagen II, collagen IX and matrilin‐3 than by growth‐related alterations in its underlying subchondral bone structure. Importantly, the present study provides a first insight into the growth‐related structural, biochemical and mechanical interaction of articular cartilage and subchondral bone. Finally, these data contribute to the general knowledge about the cooperation between the articular cartilage and subchondral bone.     

A novel blueprint for ‘top down’ differentiation defines the cervical squamocolumnar junction during development,reproductive life,and neoplasia

The cervical squamocolumnar (SC) junction is the site of a recently discovered ‘embryonic’ cell population that was proposed as the cell of origin for cervical cancer and its precursors. How this population participates in cervical remodelling and neoplasia is unclear. In the present study, we analysed the SC junction immunophenotype during pre‐ and post‐natal human and mouse development and in the adult, processes of metaplastic evolution of the SC junction, microglandular change, and early cervical neoplasia. Early in life, embryonic cervical epithelial cells were seen throughout the cervix and subsequently diminished in number to become concentrated at the SC junction in the adult. In all settings, there was a repetitive scenario in which cuboidal embryonic/SC junction cells gave rise to subjacent metaplastic basal/reserve cells with a switch from the SC junction positive to negative immunophenotype. This downward or basal (rather than upward or apical) evolution from progenitor cell to metaplastic progeny was termed reverse or ‘top down’ differentiation. A similar pattern was noted in high‐grade squamous intraepithelial lesions (HSILs), suggesting that HPV infection of the cuboidal SC junction cells initiated outgrowth of basally‐oriented neoplastic progeny. The progressive loss of the embryonic/SC junction markers occurred with ‘top down’ differentiation during development, remodelling, and early neoplasia. Interestingly, most low‐grade SILs were SC junction‐negative, implying infection of metaplastic progeny rather than the original SC junction cells. This proposed model of ‘top down’ differentiation resolves the mystery of how SC junction cells both remodel the cervix and participate in neoplasia and provides for a second population of metaplastic progeny (including basal and reserve cells), the infection of which is paradoxically less likely to produce a biologically aggressive precursor. It also provides new targets in animal models to determine why the SC junction is uniquely susceptible to carcinogenic HPV infection. Copyright © 2012 Pathological Society of Great Britain and Ireland. Published by John Wiley & Sons, Ltd.     

Recurrent HERV‐H‐Mediated 3q13.2–q13.31 Deletions Cause a Syndrome of Hypotonia and Motor,Language, and Cognitive Delays

We describe the molecular and clinical characterization of nine individuals with recurrent, 3.4‐Mb, de novo deletions of 3q13.2–q13.31 detected by chromosomal microarray analysis. All individuals have hypotonia and language and motor delays; they variably express mild to moderate cognitive delays (8/9), abnormal behavior (7/9), and autism spectrum disorders (3/9). Common facial features include downslanting palpebral fissures with epicanthal folds, a slightly bulbous nose, and relative macrocephaly. Twenty‐eight genes map to the deleted region, including four strong candidate genes, DRD3, ZBTB20, GAP43, and BOC, with important roles in neural and/or muscular development. Analysis of the breakpoint regions based on array data revealed directly oriented human endogenous retrovirus (HERV‐H) elements of ～5 kb in size and of >95% DNA sequence identity flanking the deletion. Subsequent DNA sequencing revealed different deletion breakpoints and suggested nonallelic homologous recombination (NAHR) between HERV‐H elements as a mechanism of deletion formation, analogous to HERV‐I‐flanked and NAHR‐mediated AZFa deletions. We propose that similar HERV elements may also mediate other recurrent deletion and duplication events on a genome‐wide scale. Observation of rare recurrent chromosomal events such as these deletions helps to further the understanding of mechanisms behind naturally occurring variation in the human genome and its contribution to genetic disease.     

3D imaging,registration, and analysis of the early mouse embryonic vasculature

Background: Cardiovascular development requires the input of a large number of molecular signaling molecules, and undergoes tightly regulated, three‐dimensional developmental patterning. Conventional developmental biology techniques have successfully identified many of the signaling cascades and molecular cues necessary for proper cardiovascular development, which has furnished us with a wealth of biochemical, molecular, and biologically functional information on how tightly linked cardiac and vascular development are. Still missing, however, is a genuine appreciation of the three‐dimensional (3D) nature of these important developmental steps. Results: Optical projection tomography (OPT) is a 3D imaging technique that allows for high‐resolution imaging of early mouse embryos and their developing cardiovascular systems when a PECAM‐1 antibody stain is used to highlight the vascular branching. Reported here is a method in which several 3D images of mouse embryo vasculatures can be registered, thus allowing for analysis of within‐strain variance between genetically identical mouse pups. Post‐registration, small differences in somitogenesis and ventricular trabeculation patterning can be visualized in mouse pups that differ by as little as a few hours of gestational time. Additionally, similarity metrics (cross‐correlation values) can be calculated to quantify similarities and differences. Two different mouse strains are analyzed (C57Bl/6 and CD‐1), and similar results are recognized in each strain. Conclusions: Visualizing the cardiovascular system in such a precise 3D manner allows for more accuracy in describing the steps that take place during cardiovascular development. This novel method will be applicable to many developmental biology questions in other organ systems and other species. Developmental Dynamics 242:517–528, 2013. © 2013 Wiley Periodicals, Inc.     

Muscles of Chondrichthyan Paired Appendages: Comparison With Osteichthyans,Deconstruction of the Fore–Hindlimb Serial Homology Dogma,and New Insights on the Evolution of the Vertebrate Neck

Here we present the first study comparing all the paired appendages muscles of representatives of each major extant gnathostome group. We address a crucial and enigmatic question in evolutionary and comparative anatomy: Why are the pelvic and pectoral appendages of gnathostomes, and particularly of tetrapods, in general so similar to each other? We argue that an integrative analysis of the new myological data and the information from the literature contradicts the idea that the forelimbs and hindlimbs are serial homologues. The data show that many of the strikingly similar fore‐ and hindlimb muscles of extant tetrapods evolved independently in each appendage because the ancestors of extant gnathostomes and osteichthyans only had an adductor and an abductor in each fin. Therefore, these data contradict the idea that at least some muscles present in the tetrapod fore‐ and hindlimbs were already present in some form in the first fishes with pectoral and pelvic appendages, as the result of an ancestral duplication of the paired appendages leading to a true serial homology. The origin of the pectoral girdle was instead likely related to head evolution, as illustrated by the cucullaris of gnathostomes such as chondrichthyans inserting onto both the branchial arches and pectoral girdle. Only later in evolution the cucullaris became differentiated into the levatores arcuum branchialium and protractor pectoralis, which gave rise to the amniote neck muscles trapezius and sternocleidomastoideus. These changes therefore contributed to an evolutionary trend toward a greater anatomical and functional independence of the pectoral girdle from head movements. Anat Rec, 298:513–530, 2015. © 2014 Wiley Periodicals, Inc.     

A study of the genetic variability of human respiratory syncytial virus in Croatia, 2006–2008

Human respiratory syncytial virus (HRSV) is a common etiological agent of acute lower respiratory tract disease in infants. The molecular epidemiology of HRSV in Croatia over four consecutive seasons (from 2006 to 2008) was investigated. A total of 72 HRSV samples were chosen from 696 screened cases in a pediatric clinic in Zagreb. Molecular characterization of HRSV revealed the predominance of HRSV group B viruses in the first two epidemic seasons and HRSV group A viruses in the next two seasons. According to the phylogenetic analysis, NA1 and BA9 were the predominant circulating HRSV genotypes detected during the study. Overall, 82.9% of all HRSV A strains belonged to the NA1 genotype. The HRSV B genotype BA9, detected in two consecutive seasons (2006 and 2007), was the predominant circulating HRSV B genotype, accounting for 80.6% of all HRSV B strains. This study provides data on the circulation pattern of HRSV genotypes in Croatia and their molecular characterization. J. Med. Virol. 84:1985–1992, 2012. © 2012 Wiley Periodicals, Inc.     

Clinical variability at the mild end of BRAT1-related spectrum: Evidence from two families with genotype–phenotype discordance

Biallelic mutations in the BRAT1 gene, encoding BRCA1-associated ATM activator 1, result in variable phenotypes, from rigidity and multifocal seizure syndrome, lethal neonatal to neurodevelopmental disorder, and cerebellar atrophy with or without seizures, without obvious genotype–phenotype associations. We describe two families at the mildest end of the spectrum, differing in clinical presentation despite a common genotype at the BRAT1 locus. Two siblings displayed nonprogressive congenital ataxia and shrunken cerebellum on magnetic resonance imaging. A third unrelated patient showed normal neurodevelopment, adolescence-onset seizures, and ataxia, shrunken cerebellum, and ultrastructural abnormalities on skin biopsy, representing the mildest form of NEDCAS hitherto described. Exome sequencing identified the c.638dup and the novel c.1395G>A BRAT1 variants, the latter causing exon 10 skippings. The p53-MCL test revealed normal ATM kinase activity. Our findings broaden the allelic and clinical spectrum of BRAT1-related disease, which should be suspected in presence of nonprogressive cerebellar signs, even without a neurodevelopmental disorder.